This application is a request for R24 funding through the National Center for Research Resources (NCRR) of the project entitled "Characterization of Defective Gamma-Glutamyl Carboxylase in Rambouillet Sheep." Human patients with hereditary deficiencies of gamma-glutamyl carboxylase have severe bleeding clinically, but more recently, acquired defects of carboxylation have been implicated in degenerative diseases of the aged such as vascular mineralization and osteoporosis. While hereditary deficiency of gamma-glutamyl carboxylase is uncommon, atherosclerosis and osteoporosis are significant diseases of the aged. A model of defective gamma-glutamyl carboxylation does not exist despite previous efforts to produce one through knockout mouse technology. The Specific Aims of this project are: (1) Develop and maintain a flock of sheep with defective gamma-glutamyl carboxylase activity equivalent to human gamma-glutamyl carboxylase deficiency, and provide affected lambs and tissue to investigators of gamma-glutamyl carboxylase; (2) verify that the genetic defects identified in the gamma-glutamyl carboxylase gene is responsible for diminished function, and develop a heterozygous carrier detection test for identification of carrier animals; (3) characterize the skeletal phenotype in defective gamma-glutamyl carboxylase of homozygous affected lambs; and (4) develop an in vitro gene therapy technique that may eventually be applied in vivo to this and other models of defective coagulation. Specific Aims 1, 2, and 4 will be performed at CSU under the supervision of Drs. Baker and Jennifer MacLeay, and Specific Aim 3 will be carried out at Yale University by Dr. Caren Gundberg. Preliminary studies in these sheep have identified the coagulation defect that occurs in homozygous affected lambs, and the underlying cause of this coagulopathy is markedly decreased gamma-glutamyl carboxylase activity. Genetic mutations of the gamma-glutamyl carboxylase gene have also been identified. These sheep represent the only known model of this defect in existence, but much of the underlying genetic and functional aspects of the abnormalities other than defective hemostasis are unknown. One ram and seven ewes are known heterozygous carriers of the gene. The investigators plan to identify and increase the numbers of known carrier rams to three and ewes to 24 to produce six homozygous affected lambs per year, and make rams and ewes available to investigators of gamma-glutamyl carboxylase.